Haemocytic encapsulation and the prophenoloxidase-activation pathway in the locust Schistocerca gregaria forsk

Negatively-charged Sepharose beads are not encapsulated in vivo by haemocytes of the locust Schistocerca gregaria. It has been suggested by other workers that components of the prophenoloxidase activation pathway of haemolymph might adhere to foreign surfaces and stimulate haemocyte adhesion, so one possible reason for the lack of encapsulation of beads might be due to failure of these components to adhere to the bead. Beads were thus incubated in locust haemocyte lysate supernatant, in which the prophenoloxidase pathway had been activated by Ca²⁺ or Zymosan supernatant, and were then injected into the haemocoeles of locusts. Although at least 5 proteins, including phenoloxidase, could be shown to be attached to the beads, these coated beads were not encapsulated suggesting either that the putative opsonin did not attach or that none of the components is opsonic in this system.In addition, it has been shown that the prophenoloxidase pathway in locust haemocyte lysate supernatant can be partially activated in the presence of Ca²⁺, strongly activated by β1,3-glucans and that production of phenoloxidase is not enhanced by the presence of bacterial LPS and is inhibited by a serine protease inhibitor. The changes in protein composition of unactivated and activated haemocyte lysate supernatant are discussed.     

Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.     

Influence of protease inhibitors and energy metabolism on intracellular protein breakdown in starving Escherichia coli

It has been reported that certain inhibitors of serine proteases block intracellular protein breakdown in $\text{E. coli}$ subjected to nutritional deprivation. We show here that the protease inhibitors p-toluene sulfonyl fluoride and pentamidine isethionate inhibit protein breakdown in $\text{E. coli}$ deprived of glucose, but not in bacteria starved for inorganic phosphate or ammonia. Furthermore, we find that the protease inhibitors cause a drastic decline in cellular ATP levels when glucose is omitted from the incubation medium. It is concluded that these protease inhibitors influence protein breakdown by interfering with cellular energy production, rather than by interacting with a specific serine protease.     

The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells

Experiments indicate that the prophenoloxidase activating system, which is responsible for melanin production, is also involved in immunorecognition in insects. Using haemocyte monolayer preparations of Blaberus craniifer, Galleria mellonella and Leucophae maderae, it was shown that laminarin, a β 1,3-glucan extracted from fungal cell walls and an activator of the prophenoloxidase system, enhanced the phagocytosis of test bacteria.Scanning electron microscopy of haemocyte monolayers showed that incubation of test bacteria with laminarin significantly increased the number of microorganisms attached to both the plasmatocytes and the granular cells. Furthermore with the granular cells, these bacteria became entrapped in an amorphous matrix. This material probably consists of the “sticky” proteins previously reported to be produced by crustacean haemocytes following prophenoloxidase activation. Pretreatment of haemocytes with laminarin abolished the stimulatory effect on ingestion, indicating that these “sticky” proteins are opsonic, since they would have been discharged from the haemocytes onto the glass monolayer leaving few molecules available for subsequent coating of the test particles.Preliminary biochemical studies on the G. mellonella prophenoloxidase system demonstrated that it was activated by trypsin, laminarin and laminarin G, a highly purified β 1,3-glucan, but not by dextran. Serine protease activities were also enhanced by adding laminarin to a haemocyte lysate supernatant, suggesting that the stimulatory mechanism may involve the proteolytic activity of such enzymes.     

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol.106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.     

Exocytosis of the prophenoloxidase activating system from crayfish haemocytes

Summary Lipopolysaccharides (LPS) and the -1,3-glucan laminarin G, both of which specifically activate the prophenoloxidase (proPO) activating system of crayfish haemocyte lysate, were found to induce degranulation (exocytosis) and subsequent lysis in vitro of monolayers of semigranular haemocytes from the crayfish,Pacifastacus leniusculus, (Table 1, Fig. 1 b), whereas the granular cells were unaffected (Fig. 1 c).Exocytosis of isolated semigranular or granular cells in vitro could also be evoked by the Ca² ionophore A23187 (Table 2, Fig. 1 d). In this case, the whole proPO system was released from the cellular vesicles in its inactive form, since the secreted material contained protease and prophenoloxidase as inactive proenzymes, which could be activated if LPS or -1,3-glucans were added (Table 3). The anion channel blocker SITS, which inhibits exocytosis in several systems, prevented degranulation triggered by -1,3-glucan, LPS, or ionophore.It is concluded that, in arthropods, LPS serve as an indicator of Gram negative bacteria and -1,3-glucan as an indicator of fungi. These non-self molecules elicit both the exocytotic release of the proPO system from the semigranular cells and the subsequent biochemical activation of this system.Abbreviations CFS crayfish saline - DMSO dimethyl sulfoxide - LDH lactate dehydrogenase - LPS lipopolysaccharide - proPO prophenoloxidase - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid, disodium salt     

In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes

In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%). In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems. Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes. Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated. This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase). The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

Functional analysis of plasma prophenoloxidase system in the marine mussel Perna viridis

The role of prophenoloxidase (proPO) system in recognition and phagocytosis of yeast cells by hemocytes was examined in vitro using whole plasma and proPO system isolated from the plasma of the marine mussel, Perna viridis. The proPO was isolated from the plasma by ammonium sulphate precipitation and gel filtration. The final proPO preparation was homogeneous in native PAGE, and could be activated by trypsin, α-chymotrypsin and pronase-E. Laminarin (a polymer of β-1, 3-glucan) and lipopolysaccharides (LPS) from diverse bacterial species effectively activated the isolated proPO, demonstrating the ability of this proenzyme to interact directly with microbial surface components. The susceptibility of proPO activation to inhibition by serine protease inhibitors such as soybean trypsin inhibitor (STI) or p-nitrophenyl-p′-guanidinobenzoate (p-NPGB), indicates that the isolated fraction may contain an integral serine protease domain in an inactive state. The presence of laminarin- or LPS-activated whole plasma of P. viridis facilitated adherence of yeast cells to hemocyte surface as well as eventually stimulated phagocytic uptake of the target cells by hemocytes, and no such hemocytic response was recorded with STI controls. This and other results strongly suggest that the intermediary factors generated during activation of plasma proPO system by non-self molecules play a key role in recognition and opsono-phagocytosis by hemocytes. However, the proPO system isolated from P. viridis plasma, after activation with microbial surface components, failed to show an opsonic effect.     

Isolation and purification of a cell adhesion factor from crayfish blood cells

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Soderhall and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta- 1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.     

Limulus anti-LPS factor: An anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system

Exposure of $\text{Limulus}$ amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of $\text{Tachypleus}\text{tridentatus}$ and $\text{Limulus}\text{polyphemus}$. The principle purified partially from $\text{Tachypleus}$ amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the $\text{Limulus}$ coagulation system was discussed.     

Purification and properties of rabbit reticulocyte elongation factor 1.

Elongation factor 1 has been purified about 100-fold from the lysate of rabbit reticulocytes. The native enzyme is highly asymmetric ($\text{f}\text{f}{}_0\text{= 1.53}$) and has a molecular weight of 450,000. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate shows two major bands with molecular weights of about 53,000 and 50,000. Partially purified phospholipase C and AB preparations and elastase cause dissociation of the aggregate form of the enzyme to an active form which has a molecular weight of about 50,000. The effect of these phospholipase preparations is unexplained since rabbit reticulocyte elongation factor 1 contains little or no phospholipid. A protease contamination has been considered but no evidence of protease activity has been detected in the phospholipase preparations. In aminoacyl-tRNA binding, elongation factor 1 appears to show very little, if any, turnover. However, in the presence of elongation factor 2, under conditions where polymerization occurs, elongation factor 1 functions catalytically.     

Collagenase from rabbit pulmonary alveolar macrophages.

Rabbit pulmonary alveolar macrophages produce a collagenase which lyses labeled collagen gels, specifically cleaves collagen types I, II and III, is inhibited by ethylenediaminetetraacetate, cysteine, dithiothreitol and serum but is not inhibited by a serine protease inhibitor. Alveolar macrophage collagenase activity can be enhanced by $\text{in vivo}$ BCG activation, $\text{in vitro}$ latex, silica or mycobacterium activation and by $\text{in vitro}$ uncovering of latent enzymatic activity with trypsin treatment. The production of collagenase by unactivated alveolar macrophages and the presence of “latent” collagenase in culture media of alveolar macrophages are examples of significant differences between alveolar and peritoneal macrophages.     

Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.